The goal of this proposal is to understand the role of WW domain ubiquitin-protein ligases in the regulation of renal Na v absorption via the epithelial Na v channel, ENaC. ENaC forms the pathway for Na + absorption in the kidney collecting duct and other epithelia. Thus, this channel plays a critical role in Na+ homeostasis and blood pressure control. Mechanisms that control the expression of ENaC at the cell surface play an important role in the regulation of epithelial Na v absorption. A key sequence is the PY motif, located in the C-termini of ENaC subunits. Mutation or deletion of this sequence increases the number of ENaC channels at the cell surface, causing an inherited form of hypertension (Liddle's syndrome). PY motifs mediate protein interactions by binding to WW domains, implicating a critical role for WW domain proteins. Previous work found that a WW domain protein (Nedd4) decreased Na+ current by binding to ENaC and targeting the channel for degradation. However, the role of Nedd4 in the kidney collecting duct is unknown. Moreover, it is clear that Nedd4 is part of a large family of Nedd4-related proteins. This proposal will use a systematic approach to test the hypothesis that WW domain proteins regulate ENaC-mediated Na* absorption in the collecting duct. Specific Aim 1 will identify the WW domain proteins that regulate ENaC, based on their expression patterns, their binding to ENaC subunits, and their ability to inhibit ENaC in heterologous expression systems and epithelial model systems. Specific Aim 2 will extend these results to test the hypothesis that WW domain proteins are regulated by cellular messengers (vasopressin/cAMP and Ca2+). This work will provide novel insights in the mechanisms by which these signaling pathways regulate epithelial Na + transport. Specific Aim 3 will test the hypothesis that polymorphisms in ENaC and WW domain proteins disrupt binding and/or channel regulation. By studying the functional consequences of naturally occurring genetic variation, these studies have potential to provide important new insights into the pathogenesis of renal diseases, including hypertension.